Cancer is a heterogeneous disease. A treatment should be adopted for a given type of cancer as determined by the location and genetic makeup of the tumor. However, all forms of cancer show some fundamental similarities including uncontrolled growth and self-renewal and this is in some ways driven by the pattern of gene expression. Since many different signals, regardless of cause, converge on transcription factors, and since the activation of transcription factors is a nodal point for gene transcription, transcription factors should be convergent targets for treating cancer.
Transcription factors are essential cellular components mediating different extracellular signals, including developmental and environmental, by binding to transcription responsive elements in the genome and thereby initiating the transcription of specific target genes. Aberrant transcription factor function is often associated with different diseases and leads to either increased or excessive gene transcription. As many signals and activating mechanisms converge on single transcription factors they could make efficient drug targets, e.g. for treatment of cancer.
Latent cytoplasmic transcription factors (LCTFs) are transcription factors that reside in the cytoplasm in an inactive form until they are activated through an external signal often in the form of a cell surface receptor-ligand interaction. Among these transcription factors are the family of Signal Transducer and Activator of Transcription (STAT) proteins. The STAT proteins have dual roles as they can act as both transducers of signals through the cytoplasm and function as transcription factors in the nucleus.
STAT3 is one of 6 members of the STAT family of transcriptions factors. It is an approx. 770 amino acid long protein with 6 subunits or domains; N-terminal, coiled-coil, DNA-binding, linker, SH2 and transactivation domains. STAT3 is activated by cytokine, growth factor and non-receptor mediated signaling. The canonical mechanism of STAT3 activation is kinase mediated phosphorylation of tyrosine 705 (Y705) in the SH2 domain. This triggers a reciprocal recognition of two SH2 domains of STAT3 monomers leading to the formation of a STAT3 dimer. This dimer is translocated to the nucleus, aided by importins, and transcription of target genes, through binding to DNA, is activated. On its way to the nucleus STAT3 can be further modified through serine phosphorylation, lysine acetylation or Small Ubiquitin-like Modifier (SUMO) protein attachment and these modifications serve to modulate the transcriptional activity of STAT3
STAT3 activation and dimerization through phosphorylation can be achieved through at least three responses. STAT3 can be phosphorylated by JAK kinases that are constitutively bound to cytokine receptors. Upon ligand binding, the receptors aggregate and the JAK2 proteins undergo reciprocal activation through phosphorylation and they can then recruit and activate STAT3 through binding to the SH2 domain. Alternatively growth factor receptors can directly recruit and associate with STAT3 leading to STAT3 activation through their receptor tyrosine kinase activity. Finally, non-receptor kinases, e.g. Src family kinases and Abl, can also activate STAT3. In addition non-phosphorylated STAT3 can be transported into the nucleus and participate in transcription probably by binding to other proteins to form functional heteromeric transcription factors.
In the nucleus STAT3 can interact with several other proteins including other transcription factors e.g. NF-κB.
STAT3 can also be activated by phosphorylation on serine 727 by various kinases. This phosphorylation leads to enhanced transcriptional activity. Constitutively phosphorylated serine 727 is widespread in cells from patients suffering from chronic lymphocytic leukemia (CLL).
Since STAT3 activation under normal conditions is transient, multiple negative feedback systems exist. STAT3 signaling is tightly regulated and it is not constitutively activated in normal tissue. Several endogenous negative regulators for STAT3 signaling have been found and these include Suppressor of cytokine signaling (SOCS, that bind to and inactivate JAKs) and protein inhibitor of activated STAT (PIAS). SOCS is also a gene product of STAT3 transcription demonstrating this as a negative feedback loop. Loss of PIAS or SOCS function or reduced expression will increase STAT3 activation and mutations of these regulatory factors have been found in diseases related to increase STAT3 signaling.
Finally STAT3 is dephosphorylated in the nucleus by different phosphatases and the dephophorylated STAT3 monomers are transported out of the nucleus where they once again reside latent.
The target genes of STAT3 transcription are involved in cell growth and cell cycle regulation (e.g. Cyclin Dl, c-Myc, p27), apoptosis (e.g. Mcl-1, survivin, Bcl-2, and Bcl-xL), angiogenesis (VEGF) and metastasis (e.g. MMP-2, MMP-3).
STAT3 can be activated by cytokines and growth factors including IL6, LIF, IL-10, IL-1, IL-12, EGF, TGFalpha, PDGF and G-CSF and various tyrosine and serine kinases including JAK, JAK2, JAK3, TYK2, Src, Src, Lck, Hck, Lyn, Fyn, Fgr, EGFR, ErbB-2, Grb2, JNK, P38MAPK and ERK.
STAT3 is an experimentally validated target in several cancer forms, including leukemia, lymphomas, multiple myeloma, breast cancer, prostate carcinoma, lung cancer (non-small-cell), renal cell carcinoma lung cancer, hepatocellular carcinoma, cholangiocarcinoma, ovarian carcinoma, pancreatic adenocarcinoma, melanoma, head and neck squamous cell carcinoma (Johnston, P. A; Grandis, J. R. Mol Interv. 2011 11(1): 18-26). STAT3 signaling is involved in proliferation, survival, metastasis, drug resistance and migration of cancer cells and it also links inflammation and cancer. This has been demonstrated in numerous studies in vitro, using primary cells or immortalized cell lines, or in vivo using xenograft models (cf. e.g. Sansone, P; Bromberg, J. J Clin Oncol. 2012; 30(9):1005-14, and Miklossy, G.; Hilliard, T. S.; Turkson, J. Nat Rev Drug Discov. 2013 12(8):611-29) and as such is believed to be an ideal target for cancer therapy (Yu, H.; Lee, H.; Herrmann, A.; Buettner, R.; Jove, R. Nat Rev Cancer. 2014 14(11):736-46.
The sensitivity of many cancer cell lines to STAT3 inhibition indicates an oncogene signaling dependence.
Inflammation and immunity are also important parts of cancer etiology. Cancer cells can promote inflammation in the tumor microenvironment and avoid the innate immune system. STAT3 signaling plays an important dual role in this process. STAT3 is activated by pro-inflammatory cytokine signaling and STAT3 activation opposes T-helper cell anti-tumor responses. Ablation of STAT3 signaling leads to a potent immunological antitumor response. STAT3 is more activated in tumor infiltrating immune cells than in normal tissue and targeting STAT3 causes therapeutic antitumor immunity.
In summary aberrant and deregulated STAT3 promotes cell proliferation and cell survival in both solid and hematological tumors, including breast, lung, brain, colon, prostate, lymphoma and leukemia. Direct inhibitors of STAT3 or inhibitors of STAT3 signaling are thus deemed to be able to mitigate or cure those pathological states.
The treatments for prevention, revocation or reduction of diseases like e.g. cancer are in many ways insufficient. Hence, compounds effective in modulating or inhibiting the above described STAT signaling would be desired.
The direct inhibition of STAT3 can be achieved by inhibiting the protein-protein interaction involved in STAT3 dimerization (STAT3 is a dimer of two proteins) or by blocking the protein-DNA interaction required for STAT3 binding to DNA for the initiation of transcription. Alternatively the production (biosynthesis) of STAT3 can be blocked.
The alternative to direct STAT3 inhibition is to inhibit upstream molecules in the signaling cascade responsible for STAT3 activation (e.g. the JAK kinases). The drawback with this approach is that there are multiple ways to activate STAT3.
The STAT3 SH2 has been targeted with peptidomimetics and non-peptide small molecules (e.g. S3I-M2001) to block STAT3-STAT3 dimerization and DNA binding has been blocked with oligodeoxynucleotide decoys while the production of STAT3 has been inhibited by antisense.
STAT1 is also a member of the STAT family and STAT1 is activated by either type I, type II, or type III interferon stimulation and is involved in upregulating genes that primarily are involved in controlling the growth and apoptosis of immune cells. In response to IFN-γ stimulation, STAT1 forms homodimers, or heterodimers with STAT3, that bind to the Interferon-Gamma-Activated Sequence (GAS) promoter element leading to an increased expression of Interferon-Stimulated Genes (ISG). Intact STAT1 signalling is important for the immune defense against viral and bacterial infections.
Transcription factors such as STAT3 that are disregulated in cancer and other illnesses are important targets for potential drugs but the numerous roles played by other transcription factors in healthy cells makes it important to attain transcription factor blocking drugs with a high degree of selectivity and since many transcription factors have similar activation modes and structural similarities this can be difficult to achieve.
(−)-Galiellalactone is a natural product isolated from wood-inhabiting fungi with submicromolar inhibition of IL-6/STAT3 signaling.

In U.S. Pat. No. 6,512,007 use of galiella lactone as a pharmaceutical for the treatment of e.g. inflammatory processes is disclosed.
The biological effect of (−)-galiellalactone seemingly is due to a direct inhibition of the binding of STAT3-dimers to their regulatory elements (Weidler et al in FEBS Letters 2000, 484, 1-6). Based on this proposed mechanism of action, galiellalactone has been evaluated as an anti-cancer agent. Hellsten et al reported in Prostate 68:269-280 (2008) that galiellalactone inhibits the proliferation of STAT3 expressing DU145 prostate cancer cells. Further, Hellsten et al (“Targeting STAT3 in prostate cancer: Identification of STAT3 as a direct target of the fungal metabolite galiellalactone” Nicholas Don-Doncow, Zilma Escobar, Martin Johansson, Eduardo Muñoz, Olov Sterner, Anders Bjartell, Rebecka Hellsten, AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, Oct. 19-23, 2013, Boston, Mass. Abstract nr C229; Don-Doncow, N.; Escobar, Z.; Johansson, M.; Kjellström, S.; Garcia, V.; Munoz, E.; Sterner, O.; Bjartell, A.; Hellsten, R.; J. Biol. Chem. 2014 289(23):15969-78) have shown that galiellalactone binds directly and covalently to STAT3, thus inhibiting the transcriptional activity. Galiellalactone is thus a candidate drug for treatment of cancer. Also Thaper et al (“GPA500 inhibits the STAT3 activity and suppresses ENZ-resistant Prostate Cancer in vitro”. Daksh Thaper, Sepideh Vahid, Jennifer Bishop, Martin Johansson and Amina Zoubeidi. AACR Annual Meeting 2015, Abstract nr 728) showed that galiellalactone can block STAT3 activity in enzalutamide resistant cells which leads to decreased proliferation and PSA production.
Galiellalactone also inhibits STAT1. Further, galiellalactone reduces the proliferation of non-STAT3 driven prostate cancer cells known as LNCaP cells. This clearly indicates that galiellalactone has off-target effects. As STAT1 signaling is believed to have an anti-oncogenic role in cells it would be desirable to obtain galiellalactone-based STAT3 inhibitors that have reduced STAT1 inhibitory properties. It would also be desirable to increase the anti-proliferative selectivity towards STAT3 driven cells in order to more specifically target STAT3 dependent cells.
Attempts to modify the activity and properties of galiellalactone have been reported in the art. Nussbaum et al reported in Eur. J. Org. Chem. 2004, 2783-2790 on the modification of individual functional groups of (−)-galiellalactone. Most of the resulting analogues, however, turned out to be completely inactive or much less active than (−)-galiellalactone. Especially, modifications of the conjugated double bond were reported to produce inactive compounds. WO 2012/010555 discloses the preparation and use of tricylic compounds based on a galiellalactone scaffold that inhibit STAT3 and NF-kB signaling.
Nevertheless, there remains a need for more selective inhibitors of STAT3 based on galiellalactone.